Allostreptopyrroles A–E, β-alkylpyrrole derivatives from an actinomycete Allostreptomyces sp. RD068384

Five new β-alkylpyrrole derivatives, allostreptopyrroles A–E (1–5), were isolated from the culture broth of Allostreptomyces RD068384. Their structures were elucidated by 1D and 2D NMR spectroscopic analyses, HRESIMS, and chemical derivatization. The absolute configurations of compounds 2 and 3 were predicted by comparison of experimental and calculated specific rotation data. Compounds 1–5 are the first examples of natural pyrroles substituted by formyl and carboxyl functionalities. Compounds 1, 4, and 5 showed cytotoxicity against Kasumi-1 human acute myeloblastic leukemia cells with IC50 values of 103, 105, and 105 μM, respectively, which are less active than the anticancer agent cisplatin, with an IC50 value of 70 μM.

While the pyrrole nucleus is featured in many marine natural products [6,7], pyrroles substituted with long hydrocarbon chains (pyrrole lipids) are seldomly isolated, and their presence is limited to certain marine organisms [8].A series of 3-alkylpyrrole-2-carbaldehydes/carboxylic acid/methylcarboxylate was reported from the marine sponge Oscarella lobularis (Figure 1 and Figure S54 in Supporting Information File 1) [7,9], but the actual position of the alkyl chains is very likely to be on the 5 position, as Stierle and Faulkner pointed in their study on a series of 5-alkylpyrrole-2-carbaldehydes from the sponge Laxosuberites sp.[10].From 1997 to 2017, over fifty 5-alkylpyrrole-2-carbaldehydes and 5-alkyl-2-hydroxymethylpyrroles with diversely functionalized alkyl side chains have been isolated from sponges of the genus Mycale [7,11], but no additional 3-alkylpyrroles were reported so far.

Results and Discussion
The fermentation extract of strain RD068384, cultured in A-3M medium, was fractionated on a silica gel column eluting with CHCl 3 /MeOH mixtures.Allostreptamide was obtained from the eluate with CHCl 3 /MeOH 2:1 [24].HPLC-DAD analysis of a less polar fraction, eluted with CHCl 3 /MeOH 10:1, detected several peaks with characteristic UV absorptions, which were purified by ODS flash chromatography followed by ODS HPLC to yield compounds 1-5.
Allostreptopyrrole A (1) was obtained as a greenish yellow amorphous solid.).These molecular parts accounted for four degrees of unsaturation out of five, leaving one degree for a ring structure.In addition, a highly conjugated functional group was suggested by UV maximal absorptions at 235 nm and 273 nm and HMBC correlations from the formyl and the olefinic methine protons to all sp 2 carbons except the acyl carbonyl carbon (Figure 2 and Table S1 in Supporting Information File 1).The sp 3 carbons, in contrast, constituted an alkyl chain: the six methylene units were connected in sequence to form a hexa-methylene chain as supported by overlapping six proton resonances at δ H 1.31-1.42and by inter-unit COSY and HMBC correlations.This methylene chain was blocked by an oxypropyl group, as evident from HMBC correlations from the tertiary methyl protons (δ H 1.13) to the oxygenated carbon (δ C 70.1), and one of the methylene carbons (C13: δ C 44.8).
The formyl proton H7 showed HMBC correlations to the olefinic carbons C3, C4, and C5 and the olefinic methine proton H5 was correlated with C2, C3, C4, and C7.These correlation data allowed the assignment of a carbon sequence C2-C3-C4-C5 and the attachment of the formyl group at C4. Furthermore, HMBC correlations from two methylene protons H 2 8 to the olefinic carbons C2, C3, and C4 connected the chain part at C3.A 1 H, 15 N-HMBC correlation was seen from H5 to a nitrogen at δ N 161.4,which suggested the presence of a nitrogen atom adjacent to C5.A correlation to the acyl carbonyl carbon (C6) was not available at this stage.In order to obtain further information for connectivity, compound 1 was reacted with methyl iodide and K 2 CO 3 to give a bismethylated derivative 1a.A methyl proton at δ H 3.94 was of an N-methyl group (δ C 38.5) and displayed two strong HMBC correlations to C2 and C5, which connected these carbons through a nitrogen atom to establish a pyrrole ring, and also a hydroxy group at the alkyl terminus.Another methyl proton at δ H 3.85 was of a methoxy group (δ C 51.4) and had only one HMBC correlation to C6, which provided a methoxycarbonyl (-COOMe) fragment.Finally, this fragment was placed at C2 by an HMBC correlation from H5 to C6 to complete the gross structure of 1.
Both compounds 2 and 3 were obtained as greenish yellow amorphous and their molecular formula were suggested to be the same as that of 1 from HRESITOFMS and NMR analytical data (Table 2), inferring that compounds 2 and 3 were isomers of 1.In fact, their NMR spectra were closely similar to those for 1 except a little difference in the alkyl side chain terminus.In a COSY spectrum of 2, the terminal doublet methyl proton was correlated with an oxymethine H15, which in turn was correlated with a methylene H 2 14.The pyrrole moiety with the same substituents as 1 was deduced from HMBC correlations.Therefore, compound 2 was determined to have a non-branched alkyl chain with a hydroxy group at C15.Meanwhile, 3 possessed a terminal ethyl group, which was connected to an oxymethine H14 in a COSY spectrum, thereby establishing a non-branched alkyl chain with a hydroxy group at C14.
The specific rotation values of 2 and 3 were calculated to predict their absolute configurations.For the flexible molecules 2 and 3, thousands of conformers may exist (over 52400 conformers).However, only a few are usually significantly populated (i.e., the compound exists as a rapidly equilibrating mixture of multiple conformers).In this situation, the spectroscopic properties of a molecule can be calculated as the average over the conformers, weighted according to their populations [25].The calculated specific rotations −11.4 and +16.1 were obtained for R-configured 2 and 3 from the DFT computations (see DFT methodology section), respectively, which were in good agreement with the experimentally obtained values, −6.1 for 2 and +15 for 3. Thus, R-configurations were proposed for compounds 2 and 3.However, this prediction was not confirmed by chemical derivatization due to their limited availability.
1 H and 13 C NMR spectra of compounds 4 and 5 were superimposable to those of 1 except for methylene resonances, supporting that both 4 and 5 possess the same substituted pyrrole ring and hydroxyisopropyl terminus as compound 1 (Table 3).HRESITOFMS analysis determined the molecular formula of 4 to be C 16 H 25 NO 4 and that of 5 to be C 17 H 27 NO 4 , which established that 4 and 5 are one-and two-methylene-longer congeners of 1.

Conclusion
In summary, five new alkylpyrroles, allostreptopyrroles A-E (1-5), were discovered from a fermentation extract of Allostreptomyces sp.RD068384, a strain belonging to an almost unstudied actinomycetes genus within the family Streptomycetaceae.
Compounds 1-5 are characterized by a pyrrole-2-carboxylic acid core decorated with a formyl group and an alkyl side chain.Secondary metabolites of this specific composition have not been reported.The pyrrole-2-carboxyl skeleton is a recurring framework in pyrrolic natural products including microbial pyrrolostatin and aminocoumarin antibiotics [2], plant-derived brachystemidines [26], and lamellarins from marine invertebrates [6] (Figure S55 in Supporting Information File 1).Biosynthetically, pyrrole-2-carboxylic acid is known to be derived from ʟ-proline [2].Similarly, pyrrole-2-carbaldehydes have been isolated from various natural sources including plants, marine invertebrates, and fungi [7], while 1-5 are the first to have formyl and carboxyl functionalities.Furthermore, a β-alkyl substitution is not very common in pyrrolic secondary metabolites.The most related metabolites to 1-5 are the reported alkylpyrroles from a marine sponge Oscarella lobularis [7] and pyrroloterpenes from Streptomyces [12][13][14][15], although the substitution patterns are different (Figure 1).Natural alkylpyrroles were shown to have cytotoxicity [27], antidiabetic activity [28], anti-lipid peroxidation [12], in vivo antihypoxic activity [12], and antibacterial activity [15].Though not impressive in cytotoxicity and tyrosinase-inhibitory evaluations, compounds 1-5 could be more potent in some other bioassays, which is a subject of future studies.Finally, these results supported that actinomycetes genera with little or no chemical study are a fruitful reservoir for discovering new natural molecules.

Methylation of 1
Allostreptopyrrole A (1, 2.0 mg, 0.007 mmol) and K 2 CO 3 (4.4mg, 0.032 mmol) were stirred in dry DMF (0.5 mL) at 50 °C for 10 min.Methyl iodide (19 μL, 0.32 mmol) was added and the mixture was stirred at this temperature for 12 h [29].Reaction completion was monitored by TLC.The solution was diluted with water and extracted with EtOAc three times.The organic layer was washed with brine and evaporated to dryness to afford bismethylated derivative of 1 (1a, 1.9 mg, 88% yield).

DFT methodology
Prior to the calculations of the molecular properties of compounds 2 and 3, their conformational ensembles, datasets of a structure and population of each conformer [25], were determined using a Spartan 24 parallel package conformational search tool (Wavefunction Inc, USA), followed by geometry optimization of the most weighted conformers (Boltzmann distribution weight lower to 0.004).Eleven and seven weighted conformers for compounds 2 and 3 were obtained, respectively, each forming equilibrium mixtures.The quantum chemical method for the calculation of conformer distribution was ωB97X-V/6-311+G(2df,2p)[6-311G*] [30].The CPCM solvation model for methanol was used [31].Range-separated hybrid GGA (RSH-GGA) functional, including dispersive interaction with 6-31G* as the polarization basis set (ωB97X-D/6-31G* method), was used for energy and geometry optimization [32].

Bioactivity
Cytotoxicity and tyrosinase assays were carried out according to the procedures previously described [33,34].The detailed procedures are available in Supporting Information File 1.

Table 1 : 1 H and 13 C NMR data for 1 and 1a. a 1 1 b 1a
a NMR data were recorded in CD 3 COCD 3 at 500 and 125 MHz for 1 H and 13 C, respectively.bRecorded in CD 3 OD.cAssigned from HMBC.d Interchangeable.eAssignedfrom COSY.f Overlapping signals read from HSQC.g15 N chemical shift determined from15N HMBC.

Table 2 :
1H and13C NMR data for compounds 2 and 3 in CD 3 COCD 3 .23Positionδ C δ H, mult, J in Hz a Interchangeable.b Assigned from COSY.c Overlapping signals read from HSQC.